Ergothioneine (Egt) is a rare sulfur-containing amino acid widely distributed in edible fungi and bacteria. As a natural antioxidant, Egt has demonstrated significant application potential in the food, pharmaceutical, and cosmetic industries. However, conventional Egt production predominantly relies on chemical synthesis and biological extraction methods, which suffer from low titers and cannot meet the escalating market demand. With advances in synthetic biology, genetic engineering of suitable microbial chassis strains coupled with fermentation process optimization has emerged as a research hotspot for enhancing Egt biosynthesis efficiency. This review systematically examines Egt's applications, metabolic pathways, microbial synthesis strategies, and fermentation optimization approaches, while also prospecting future research directions and technical challenges in Egt production.